1. Field of the Disclosure
The invention relates to the conversion of prothrombin present in a blood fluid into thrombin, and more particularly to an apparatus and method for increasing the concentration of activated cascade coagulation proteins in blood.
2. Description of the State of the Art
Blood plasma coagulation is thought to occur through a series of interconnected self-amplifying, zymogen-enzyme conversions (FIG. 1) that penultimately produce thrombin (FIIa), a powerful serine protease. Dubbed the “coagulation cascade”, in the final steps, prothrombin is converted to thrombin by a multi-protein complex with calcium called the prothrombinase enzyme complex. Thrombin is an enzyme that hydrolyses fibrinogen into fibrin units that polymerize into a fine mesh, which, in turn, causes plasma to form a gel or clot. Thrombin is a serine protease of the trypsin family with a molecular weight of approximately 34 kDa. It consists of two polypeptide chains.
The coagulation cascade is usually divided into two branches for convenience of discussion and coagulopathy testing. The intrinsic and extrinsic branches can be separately potentiated but merge into a common pathway leading to thrombin. The extrinsic pathway is responsible for hemostatic control and response to vascular injury. The intrinsic pathway starts when blood comes into contact with procoagulant materials, causing Factor XII activation. The contact activation of blood plasma coagulation by procoagulant materials has been well studied as in J Biomed Mater Res. 1995 August; 29(8):1005-16 and J Biomed Mater Res. 1995 August; 29(8):1017-28, the entire contents of which are incorporated herein by reference.
Procoagulant refers to any element or activity that causes the blood fluid to form a thrombin mediated fibrin clot. Procoagulant agents include a variety of negatively charged surfaces, including organic and inorganic materials including kaolin, cotton, ceramic, glass, ellagic acid and the like. Procoagulant agents are known to be used singularly or in combination.
Binding to these negatively charged surfaces induces a conformational change in Factor XII (FXII) that allows it to proteolytically activate prekallikrein (PK). PK proteolytically activates FXII, producing a positive feedback loop that amplifies the system and leads to activation of FXI and cleavage of high-molecular-weight kininogen (HK) by kallikrein.
A subsequent product of Factor XIIa activation by procoagulant materials is the formation of the prothrombinase enzyme complex that consists of the serine protease, Factor Xa, and the protein cofactor, Factor Va. The complex assembles on negatively charged phospholipid membranes in the presence of calcium ions. The addition of calcium salt to citrate anti-coagulated blood fluids to enable the coagulation cascade to proceed is often specifically referred to as recalcification. The prothrombinase enzyme complex catalyzes the conversion of prothrombin (Factor II), an inactive zymogen, to thrombin (Factor IIa), an active serine protease. Although it has been shown that Factor Xa can activate prothrombin when unassociated with the prothrombinase enzyme complex, the rate of thrombin formation is severely decreased under such circumstances.
The important blood factors involved in coagulation that circulate in normal blood are present in much lower concentration than a plethora of other blood proteins (about 490 of them at concentrations varying over six decades). Among many unresolved issues, one is the manner in how high-concentration blood proteins such as albumin, fibrinogen, or IgG fail to compete with assemblage of activation-complex proteins at procoagulant surfaces that are composed of proteins at considerably lower blood concentrations (referred to an “adsorption-dilution” effect). Zhuo et al., Biomaterials. 2007 October; 28(30): 4355-4369, the entire contents of which are incorporated herein by reference, discloses the rate of FXIIa accumulation in whole-plasma is found to decrease with time in the continuous presence of activating surfaces, leading to a steady-state FXIIa yield dependent on the initial FXII solution concentration for procoagulant particles suspended in plasma. The authors explain that the results strongly suggest that activation competes with an autoinhibition reaction in which FXIIa itself inhibits FXII→FXIIa. Erwin A. Vogler and Christopher A. Siedlecki, Biomaterials. 2009 April; 30(10): 1857-1869, the entire contents of which are incorporated herein by reference, have comprehensively reviewed the additional uncertainties that exist in contact activation coagulation chemistries including autoactivation, autohydrolysis, and autoinhibition reactions. See generally, FIG. 1.
Prothrombinase enzyme complex assembly begins with the binding of Factor Xa and Factor Va to negatively charged phospholipids on plasma membranes. Once bound to the plasma membrane, Factor Xa and Factor Va rapidly associate in a 1:1 stoichiometric ratio to form the prothrombinase enzyme complex. The assembly of the prothrombinase enzyme complex is calcium dependent. The fully assembled prothrombinase enzyme complex catalyzes the conversion of the zymogen prothrombin to the serine protease thrombin. When associated with the prothrombinase enzyme complex, the catalytic efficiency of Factor Xa is increased 300,000-fold.
The coagulation system is under extraordinarily tight regulation by both stoichiometric and dynamic inhibition systems. The concentrations of plasma procoagulants, the stoichiometric inhibitors, and the constituents of the dynamic inhibition processes largely regulate the ultimate amount of thrombin produced. For example, Factor Va of the prothrombinase enzyme complex is inactivated following cleavage by activated protein C, which reduces the ability of Factor V to bind to Factor Xa. Factor Xa of the prothrombinase enzyme complex is inhibited by the antithrombin III, which also acts to inhibit thrombin. Thrombin in plasma is only transiently stable having a half-life of approximately 10-15 seconds largely through the inhibitory properties of the plasma protein antithrombin III (Advanced Engineering Materials Volume 11, Issue 12, pages B251-B260, December, 2009, the entire contents of which are incorporated herein by reference.)
The exact chemistry of autoactivation, autohydrolysis, and autoinhibition reactions of the coagulation system remain unknown. Resolution of an improved reaction scheme for contact activation may require a solution to vexing problems of protein adsorption and protein-adsorption competition, as well as a greatly improved understanding of the biochemistry involved in surface activation of zymogens.
The role of thrombin in the coagulation cascade and its use for bioengineering applications of fibrin gels has been reviewed, for example by Janmey et al., J. R. Soc. Interface (2009) 10, 1-10, the entire contents of which are incorporated herein by reference. Thrombin is used clinically to control bleeding during surgery, for burns and in certain trauma situations. Bovine thrombin is also a component of some commercial tissue glues.
Conventional commercial thrombin therapeutics are purified from pooled human and animal blood products and as such run the risk of contamination with viruses such as the HIV and hepatitis viruses. In comparing three commercial thrombin preparations, Suzuki and Sakuragawa found that the preparations contained contaminating proteins, and the human preparation contained immunoglobulin G, hepatitis B surface antigen antibodies and human immunodeficiency antibodies. Xenogeneic immunization with bovine thrombin has been reported in patients who have developed self-reactive antibodies to both human thrombin and human factor V (factor V is a contaminant in the bovine thrombin preparation). In addition, concerns have recently been raised regarding the possible contamination of bovine products with pathogens such as the bovine spongiform encephalitis agent, which is not detectable or inactivated by conventional means. Therapeutic human blood products are also subject to contamination by viral particles such as the hepatitis virus and the human immunodeficiency virus. There are also cultural and religious reasons that bovine thrombin is not found to be acceptable for clinical use.
Recombinant thrombin has recently been approved by the FDA and is being promoted commercially as an alternative to bovine plasma-based thrombin, which can potentially cause the formation of inhibitory antibodies to bovine thrombin, and other safety concerns associated with an animal-derived blood product. However, recombinant thrombin has also been demonstrated to have immunogenicity issues in some patients who received the product.
It has long been understood, however, that the safest condition for a surgical patient in need of a fibrin sealant would result from a two component biological sealant preparation in which the thrombin component would be harvested from the same donor in which the fibrinogen protein component was harvested-forming a fully autologous biological sealant or glue so as to avoid any risk of blood borne disease transmission.
The concept of utilizing thrombin and/or fibrinogen sourced from the patient in a medical procedure performed on that patient dates to 1974 and is not novel. Cederholm-Williams PCT Patent (WO94/00566-10 Jan. 1994) describes an improved fibrin glue in which the thrombin component, which required thirteen steps, including centrifugation, and separation of intermediate precipitates and adjusting the ionic strength of the blood and pH of the plasma to prepare, would be combined with a fibrinogen component also sourced from the plasma of the same donor. However, these many preparation steps are so time consuming they become impractical for use in the perioperative theater where processing times should be less than one hour.
Three years later, in 1997, Hirsh, et al. (U.S. Pat. No. 5,643,192) follows Cederholm-Williams by teaching another method of preparing fibrin glue in which both the fibrinogen and thrombin components of a fibrin glue are sourced from the same donor's plasma. The Hirsh patent describes a method of preparing thrombin in which most of the fibrinogen in the plasma is first precipitated and removed to prepare a supernatant and then clotting the residual fibrinogen in the supernatant which is different and simpler than the method taught by Cederholm-Williams, but does not result in a commercially useful thrombin in that the thrombin provides clotting speeds of five seconds or less for only 4 minutes, and less than 10 seconds for only 47 minutes.
These clotting speeds are unsuitable to the needs of surgeons who could not plan their entire surgeries around the limitations of the Hirsh, et al. fibrin glue.
Surgeons predominately require a fast acting clotting time (<5 seconds) for hemostasis and tissue sealing or adhesion. Slow clotting biological glues (>5 seconds) will often be transported away from the wound site by oozing and bleeding before they can perform their function. A surgeon utilizing the Hirsh fibrin glue would be required to arrange his surgery so that the hemostasis and tissue sealing intended for treatment with the Hirsh fibrin glue would occur within the 4 minute window where the clotting time was less than 5 seconds, making the Hirsh invention totally impractical for most surgeries which predominantly require rapid hemostasis and tissue adhesion throughout the surgery, the time span of which could extend to six hours.
Sternberger discloses in Br J Exp Pathol. 1947 June; 28(3): 168-177, the entire contents of which are incorporated herein by reference, that ethanol can be used to stabilize thrombin activity in plasma. Coelho et al. in U.S. Pat. No. 7,056,722, the entire contents of which are incorporated herein by reference, disclose an invention for a simple, practical, and fast method of preparing stable human thrombin from a donor's blood using ethanol as a thrombin stabilizing agent. The method provides fast clots (less than 5 seconds) that are stable throughout a lengthy surgery (e.g., six hours) by the addition of ethanol at a concentration of 8% to 18%. Coelho et al. further teaches that an apparatus for manufacturing a thrombin preparation from blood that is not stabilized with ethanol is totally impractical for the broad range of surgeries in which thrombin is used. The inventors had to identify the very narrow range of ethanol concentration that was sufficiently low to avoid inhibition of the coagulation cascade which would prevent generation of the thrombin but sufficiently high enough to stabilize thrombin.
Kumar et al. disclose in JECT 2005; 37:390-395 and in JECT. 2007; 39:18-23, the entire contents of which are incorporated herein by reference, teaches that the easiest way to initiate thrombin production is to add calcium ions to citrated plasma. Here, the surplus calcium allows the clotting cascade to initiate and thrombin to be produced. Kumar further teaches that the disadvantage with this procedure is that the stability of the thrombin activity in the produced thrombin is short, and because of the inhibition of the coagulation cascade by Protein S, Protein C and anti-thrombin III, the activity is typically decreased to a nonfunctional state within 20 minutes of production. Kumar et al. further disclose a method and device to circumvent these limitations in which a stable thrombin product may be produced when the inhibitory enzymes are partially inactivated using ethanol. To concentrate and activate thrombin, a mixture of calcium chloride and ethanol is added to citrate anti-coagulated plasma in the presence of a negatively charged surface. The negatively charged surface initiates the formation of the prothrombin-FV-FXa complex, whereas the mixture of calcium chloride and ethanol (the thrombin reagent) provides the chemical constituents to inactivate inhibitors of thrombin and to partially stabilize the thrombin so that it can be used hours after production. Kumar and Chapman in JECT. 2007; 39:18-23, the entire contents of which are incorporated herein by reference, also disclose a method to generate autologous human thrombin from whole blood instead of plasma as the starting source biologic fluid within a 30-minute period, however, like much of the other prior art, it still employs ethanol as an additive to stabilize the thrombin product however with thrombin activity continuously decaying over time even with storage at 4° C.
Thrombin serum/ethanol preparations such as those disclosed in Coelho et al. in U.S. Pat. No. 7,056,722 and Kumar are not biocompatible solutions as disclosed by Semple et al. in J Oral Maxillofacial Surg 66:632-638, 2008, the entire contents of which are incorporated herein by reference. The preparations are cytotoxic unless substantially diluted prior to administration to a final ethanol concentration of less than 4%.
The use of ethanol as a stabilizer for thrombin is also disclosed in McGinnis et al. in U.S. Pat. Pub. 2004/0120942, the entire contents of which are incorporated herein by reference. McGinnis et al. also disclose the use of “contact activation agents,” which are meant to be agents involved in the intrinsic pathway of coagulation, and includes but is not limited to glass, glass beads, diatomaceous earth, ceramics, kaolin and any combination thereof.
Kanayinkal et al. in U.S. Pat Pub. 2009/0044852, the entire contents of which are incorporated herein by reference, disclose contacting the thrombin composition with a stabilizing agent to provide a thrombin composition having a stable-life of more than about 10 hours wherein the stabilizing agent comprises ethanol in a range of 8% to 25%, a polyol, PEG, ammonium sulfate, a non-polar solvent, a polar solvent, a methyl isobutyl ketone alcohol, glycol, tricloroacetic acid, acetate salt, or any combination thereof. While this system does provide an extended-life thrombin producing composition, biocompatibility complications may be introduced due to the presence of the stabilizing agent.
Nowakowski in U.S. Pat. No. 6,159,232 issued on Dec. 12, 2000; in U.S. Pat. No. 6,478,808 issued on Nov. 12, 2002; in U.S. Pat. No. 6,482,223 issued on Nov. 19, 2002 and U.S. Pat. No. 6,989,022 issued on Jan. 4, 2006 and in US Pat. Pub. No. 2006/0178610 published on Aug. 10, 2006, the entire contents of all of which are incorporated herein by reference, discloses a wound closure method and apparatus wherein the clotting cascade of a blood fluid is first initialized while the fluid is outside the body and within a substantially enclosed sterile container. The clotting cascade initiation apparatus may include a procoagulating agent (a component capable of causing blood fluid to form a clot), mechanisms to substantially neutralize an anticoagulant (such as adding liquid protamine sulfate to the blood fluid so as to inhibit the anticoagulant heparin), or a mechanism to substantially neutralize an anticlot (examples of anticlot inhibitors are described as tranexamic acid and plasminogen binding material). The clot-activated blood fluid is then deposited about the wound wherein the clotting continues.
Recently, the importance of thrombin beyond its key role in the clotting process has been investigated. Bae et al. in J Cell Physiol. 2009; 219(3): 744-751, the entire contents of which are incorporated herein by reference, reviews that thrombin, in addition to playing a central role in the formation of blood clots by cleaving fibrinogen to fibrin, possesses diverse biological activities related to inflammation, allergy, tumor growth, metastasis, apoptosis, and tissue remodeling. Thrombin's ability to modulate a variety of cell functions is achieved in m any cases through the interaction with specific cell surface receptors. All of the known thrombin receptors belong to the protease-activated receptor (PAR) family and are characterized by a peculiar proteolytic mechanism of activation. Receptor activation occurs when thrombin cleaves the extracellular domain of the receptor exposing a tethered ligand. Among the receptors of the PAR family, thrombin can interact specifically with PAR-1, -3, and -4. Additionally thrombin is a powerful mitogenic agent for some cells.
Despite this progress, the ideal method of manufacturing single donor thrombin serum at the point of care remains lacking. The ideal thrombin preparation apparatus and method must be safe to use within the body and should therefore be derived from the patient's own blood and should not require cytotoxic additives such as ethanol to overcome thrombin stability issues.
The above prior art reflects the state of the art of which applicant is aware and is included herewith to discharge applicant's acknowledged duty to disclose relevant prior art. It is stipulated, however, that none of these references teach singly nor render obvious when considered in any conceivable combination the nexus of the instant invention as disclosed in greater detail hereinafter and as particularly claimed.